Identification of risk factors and mosquito vectors associated with dengue virus infection in American Samoa, 2017

Introduction The first outbreak of dengue in American Samoa was reported in 1911. Sporadic outbreaks have been reported since, as were outbreaks of other pathogens transmitted by Aedes species mosquitoes including Ross River, chikungunya, and Zika viruses. During an outbreak of dengue virus-type 2 (DENV-2) in 2016–2018, we conducted household-based cluster investigations to identify population-specific risk factors associated with infection and performed entomologic surveillance to determine the relative abundance of Ae. aegypti and Ae. polynesiensis. Methods and findings We contacted dengue patients who had tested positive for DENV infection and offered them as well as their household members participation in household-based cluster investigations. For those that accepted participation, we also offered participation to residents of households within a 50-meter radius of each case-patient’s home. Questionnaires were administered and serum specimens collected for testing by RT-PCR and anti-DENV IgM ELISA. Adult female mosquitoes were aspirated from inside and outside participating households and tested by RT-PCR. We analyzed characteristics associated with DENV infection in bivariate analyses. A total of 226 participants was enrolled from 91 households in 20 clusters. Median age of participants was 34 years (range: <1–94), and 56.2% were female. In total, 7 (3.2%) participants had evidence of DENV infection by IgM ELISA (n = 5) or RT-PCR (n = 2). Factors significantly associated with DENV infection were reporting a febrile illness in the past three months (prevalence ratio: 7.5 [95% confidence interval: 1.9–29.8]) and having a household septic tank (Fisher’s Exact Test, p = 0.004). Of 93 Ae. aegypti and 90 Ae. polynesiensis females collected, 90% of Ae. aegypti were collected inside homes whereas 83% of Ae. polynesiensis were collected outside homes. DENV nucleic acid was not detected in any mosquito pools. Sequencing of the DENV-2 from patient specimens identified the Cosmopolitan genotype of DENV-2 and was most closely related to virus detected in the Solomon Islands during 2016. Conclusions This investigation demonstrated that dengue is a continuing risk in American Samoa. Increased frequency of infection among residents with a septic tank suggests a need to investigate whether septic tanks serve as larval habitats for mosquito vectors of DENV in American Samoa. Future efforts should also evaluate the role of Ae. polynesiensis in DENV transmission in the wild.


Introduction
The first outbreak of dengue in American Samoa was reported in 1911. Sporadic outbreaks have been reported since, as were outbreaks of other pathogens transmitted by Aedes species mosquitoes including Ross River, chikungunya, and Zika viruses. During an outbreak of dengue virus-type 2 (DENV-2) in 2016-2018, we conducted household-based cluster investigations to identify population-specific risk factors associated with infection and performed entomologic surveillance to determine the relative abundance of Ae. aegypti and Ae. polynesiensis.

Methods and findings
We contacted dengue patients who had tested positive for DENV infection and offered them as well as their household members participation in household-based cluster investigations. For those that accepted participation, we also offered participation to residents of households within a 50-meter radius of each case-patient's home. Questionnaires were administered and serum specimens collected for testing by RT-PCR and anti-DENV IgM ELISA. Adult female mosquitoes were aspirated from inside and outside participating households and tested by RT-PCR. We analyzed characteristics associated with DENV infection in bivariate analyses. A total of 226 participants was enrolled from 91 households in 20 clusters. Median age of participants was 34 years (range: <1-94), and 56.2% were female. In total, 7 (3.2%) participants had evidence of DENV infection by IgM ELISA (n = 5) or RT-PCR (n = 2). Factors significantly associated with DENV infection were reporting a febrile illness in the past three months (prevalence ratio: 7.5 [95% confidence interval: 1.9-29.8]) and having a household septic tank (Fisher's Exact Test, p = 0.004). Of 93 Ae. aegypti and 90 Ae.

Introduction
Dengue is the most common mosquito-borne viral disease worldwide and a leading cause of morbidity throughout the tropics, where an estimated 50 million symptomatic infections occur each year [1][2][3][4]. Following infection with any of the four mosquito-transmitted dengue virus-types (DENV-1-4), illness can range from a mild, nonspecific acute febrile illness (AFI) to severe dengue characterized by increased intravascular permeability leading to shock, and hemorrhage [1]. American Samoa is a territory of the United States (U.S.) located in the South Pacific Ocean. Multiple reports of dengue outbreaks were made from American Samoa during 1911-1945 [5], and DENV was first detected by the U.S. Navy in 1972 [6]. Soon after, an epidemic of Ross River virus occurred throughout the South Pacific, including American Samoa during 1979-1980, in which Ae. polynesiensis was implicated as a major vector [7,8]. American Samoa has since experienced outbreaks of all four DENVs, and by 2010 more than 95% of adults were estimated to have been infected with at least one DENV [9]. Consistent with global trends in the emergence of other viruses transmitted by Aedes aegypti, outbreaks of chikungunya and Zika viruses occurred in American Samoa in 2014 and 2016, respectively [10,11].
Due to trends in human movement and the limited flight range of peri-domestic Ae. aegypti mosquitoes, dengue cases typically cluster around households and areas where people congregate (e.g., schools, churches) [12,13]; however, in American Samoa Ae. polynesiensis are more prevalent than Ae. aegypti [14,15]. Ae. polynesiensis has long been known to be an effective DENV vector in the laboratory [16], although it has yet to be incriminated as a DENV vector in the wild [17]. Hence, the predominance of Ae. polynesiensis in American Samoa may result in differences in risk factors for DENV transmission compared to areas where Ae. aegypti is the dominant or sole vector. Ae. polynesiensis females tend to feed and rest outdoors more than Ae. aegypti females, which frequently enter houses and other buildings [18][19][20][21]. Both species have limited flight ranges [18,22,23], and both mainly utilize water-holding natural and artificial containers for larval development in American Samoa [14,15]. In some settings, Ae. aegypti also develop in septic tanks or other underground water habitats [24][25][26][27].
During an outbreak of DENV-2 in American Samoa that started in 2016 [28], we sought to determine the incidence of DENV infection among household contacts of cases, identify risk factors associated with DENV infection, and characterize the distribution of known and suspected mosquito vectors of DENV. To do so, we conducted household-based cluster investigations that included collection of epidemiologic as well as entomologic data.

Ethics statement
As this investigation was activity designed to identify, characterize, and control an immediate public health threat, following CDC Human Subjects Review it did not meet the definition of research and IRB review was not requested. Adults aged 18 years or older provided written consent for themselves and children aged <18 years for whom they were responsible. All minors provided assent if able. Consent, assent, and questionnaires were conducted in Samoan or English. Data were not anonymized prior to analysis, but were deidentified.

Setting
American Samoa is located in the South Pacific Ocean, west of the Cook Islands and north of Tonga. It forms the eastern portion of the Samoan archipelago, while the nearby islands to the west comprise the independent country of Samoa. In 2010, the population of American Samoa was 55,519 [29], most (>95%) of which lived on the main island of Tutuila. Median age in American Samoa in 2010 was 22 years, and more than one-third of the population was aged <16 years. Most (92%) residents were Pacific Islander, and average household size was 5.6 people. Total land area of American Samoa is 76.8 square miles (199 square kilometers).

Household-based cluster investigations
Household cluster investigations were conducted during September 25-October 3, 2017.
Investigation teams consisted of an interviewer, phlebotomist, and Environmental Health Service Officer. All cluster investigations were conducted on Tutuila, where all reported dengue cases resided. Patients reported to the American Samoa Department of Health (ASDOH) with suspected dengue were eligible to be contacted for this investigation if they had tested positive for DENV infection in the 30 days prior to the date of report to ASDOH by detection of: 1) DENV non-structural protein-1 (NS1) by rapid diagnostic test (SD BIOLINE Dengue Duo, Abbott Laboratories, Chicago, IL) performed in American Samoa; or 2) DENV nucleic acid by reverse transcription polymerase chain reaction (RT-PCR) [30] performed at the Hawaii State Laboratory in Honolulu, HI. Eligible case-patients or their parent or guardian were contacted by telephone to orient them to the investigation. If interested in participating, household visits were scheduled during which participation was offered to all residents of the case-patient's household. In addition, on the same day as the household visit to the casepatient's household, teams walked door-to-door to visit all households within a 50-meter radius of the case-patient's household, orient all available heads-of-household to the investigation, and offer participation to them and other household members. Households were not replaced or revisited if contact could not be made with the head-of-household or if they chose not to participate in the investigation. While index case-patients were invited to participate, cluster investigations were still conducted if case-patients were unavailable to be offered or declined participation.
Among participating households, adult heads-of-household were asked to complete a questionnaire regarding household-level characteristics including housing structure, mosquito control methods, presence of screens on windows and doors, air conditioning, income, and presence of a septic tank. An individual questionnaire was administered to all participating household members, which collected information on recent febrile illness, medical and travel history, mosquito avoidance behaviors, time spent at home, and education level. Parents or guardians answered questionnaires by proxy for participants aged <8 years. Educational materials about dengue, the need to seek medical care for febrile illness, and recommended approaches to prevention were provided to each household regardless of participation in the investigation.
Blood specimens were collected from each participant and stored at 4˚C until serum could be separated, after which serum was frozen at -70˚C and transported to CDC Dengue Branch in San Juan, Puerto Rico. All specimens were tested by Trioplex RT-PCR to detect DENV, CHIKV, and ZIKV nucleic acid [30] as well as anti-DENV IgM using a commercially available diagnostic assay (DENV Detect IgM Capture ELISA, InBios International, Inc, Seattle, WA).

Entomologic surveys
Among households that provided consent for mosquito sampling, a Prokopack aspirator [31] was used to collect mosquitoes inside the house and from around the outside of the house starting from the immediate periphery of the house and working outward, each for 15 minutes. The inside and outside of the houses were surveyed for potential water-holding containers and scored if the container held water and the presence/absence of mosquito larvae or pupae. Fewer containers were found indoors, which allowed identification of immature mosquitoes to sub-genus, whereas this was not logistically feasible for outdoor containers.
The outdoor inspections for containers and immature mosquitoes covered the entire household yard, usually clearly delimited by hedgerows, driveways, roads, streams, trees, rocks, etc. Aspirated adult mosquitoes were transported in a cooler to the laboratory where they were identified to species using the taxonomic keys of Ramalingam and Huang [32,33]. Ae. aegypti and Ae. polynesiensis females were sorted into pools by species, house, and indoor/ outdoor collection. Pools containing 1-18 females per vial were stored in RNAlater at -20˚C until shipped to CDC Dengue Branch in Puerto Rico for testing by Trioplex RT-PCR [30].

Definitions
"Index cases" were defined as the reported, laboratory-positive case-patients with dengue that initiated each cluster investigation. "Clusters" were comprised of all households within a 50-meter radius of the index patients' household and included vacant and occupied homes. "Participants" were household members who answered the individual questionnaire and provided a serum specimen. Participants of household-based cluster investigations were defined as being "laboratory-positive" if their serum specimen tested positive for DENV infection by either RT-PCR or anti-DENV IgM ELISA. Participants that tested negative by both assays were defined as being "laboratory-negative."

Data analyses
All data cleaning and analyses were performed using SAS 9.4 (SAS Institute Inc., Cary, NC). To avoid sampling bias, two index case-patients that participated in cluster investigations were excluded from analyses to identify risk factors associated with DENV infection. Generalized estimating equation (GEE) analyses assuming an exchangeable correlation matrix (equal correlations among dependent observations) with a Poisson distribution (logarithm link) were used to model bivariate associations (prevalence ratios) among individual and household characteristics, entomological factors, and the outcomes of DENV infection. The GEE method accounts for correlations in data of participants from the same household and cluster that might otherwise bias variance estimates. Confidence intervals were constructed using robust estimates for standard errors. Fischer's Exact Test was used for small cell sizes. Due to small number of participants with DENV infection, a multivariate model of characteristics associated with DENV infection was not performed.
The attributable symptomatic infection rate was calculated by subtracting the percentage of participants without evidence of DENV infection that reported an acute febrile illness in the past three months from the percentage of participants with evidence of DENV infection that reported an acute febrile illness in the past three months.
For entomologic data, descriptive statistics were calculated to characterize the frequency with which mosquito species were detected in and around homes and the various water-containers that were observed to be colonized.

Molecular epidemiology
Specimens from dengue case-patients identified during the 2016-2018 outbreak were forwarded to CDC Dengue Branch for molecular phylogenetic analysis. The DENV-2 envelope glycoprotein (E) gene from 10 specimens amplified in tissue culture was sequenced using a Sanger bi-directional method described previously [34]. Briefly, primers specific for DENV-2 were used to amplify the E gene by RT-PCR. The amplification product was purified and sequenced with eight sequencing reactions on an ABI3500 Genetic Analyzer instrument (ThermoFisher). Genotyping analysis was performed by phylogenetic comparison of the 10 complete E gene sequences (1,485 basepairs) obtained in this study with 80 additional reference sequences retrieved from GenBank representing various DENV-2 genotypes. Sequences were aligned using MAFFT and a Bayesian phylogenetic tree was reconstructed using the BEAST v1.8.4 package [35,36]. All sequences obtained in this investigation were published in GenBank (accession numbers MK244386-MK244395).

Household cluster investigations
A total of 21 household-based cluster investigations were conducted around the residences of 21 index cases. Of 142 housing structures in all clusters, ten (7.0%) were vacant. Of the 132 occupied households, a head-of-household from 98 (74.2%) was offered participation in the investigation, of whom 97 (99.0%) accepted. Of 573 residents of all participating households, 252 (44.0%) were either not available or declined participation. Of the remaining 321 residents, 228 (71.0%) participants both completed the survey and provided a serum specimen, including two index case-patients that were removed from additional analyses. Among 226 participants that were included in the analysis, median age of participants was 35 years (range: <1-94), and more than half (56.2%) were female.
A total of seven (3.1%) participants tested laboratory-positive for DENV infection, of which two tested positive by RT-PCR and five by IgM ELISA. No participants tested positive for infection with CHIKV or ZIKV. Participants laboratory-positive for DENV infection resided in four (20.0%) of the 20 clusters and six (6.6%) of the 91 households.
Of 56 (24.8%) participants that reported a febrile illness in the past three months, seven (12.1%) were laboratory-positive for DENV infection. Febrile illness was reported among five (71.4%) participants that were laboratory-positive for DENV infection, and 51 (23.3%) that were laboratory-negative for DENV infection. The percentage of febrile illness attributable to DENV infection was 48.1%.
Following bivariate analysis, factors not significantly associated with DENV infection were age, sex, mosquito bite frequency, use of repellents, time spent at home, and use of a bed net ( Table 1). The sole individual characteristic associated with DENV infection was reporting a febrile illness in the prior three months. Household characteristics associated with DENV infection included presence of a septic tank (Table 2). Although only four participants reported having burned citronella candles to attempt to control household mosquitoes, one tested positive for DENV infection, resulting in this uncommon behavior being significantly associated with DENV infection.

Entomologic findings
From 90 unique premises that were inspected, we identified 552 outdoor containers with potential to become aquatic habitats for immature mosquitoes (Fig 1). The most common (n = 128, 23%) were plastic containers including cups, lids, and bags. Among the categories of (100) 0 (0) -Abbreviations: DENV = dengue virus; PR = prevalence ratio; CI = confidence interval 1 Education level was missing for 2 participants who were DENV negative. 2 Univariable models were performed for participants who reported being bitten in the evening versus those that did not report being bitten in the evenings. 3 Univariable models were performed for participants who reported being bitten at home versus those that did not report being bitten at home.
https://doi.org/10.1371/journal.pgph.0001604.t001 containers surveyed, tires, buckets, and coconuts were the most common, representing a combined 27% of identified containers and 32% of containers that were producing pupae. We found water-holding containers inside 26 (31%) of the 83 houses inspected. The most common indoor container was used to catch water underneath faulty plumbing, which was found in 19 of the households, five of which contained larvae or pupae of Stegomyia species. Other indoor containers included those used to root plant cuttings, collect water from leaking roofs, ant guards under table legs, and miscellaneous containers. Of these, immature Stegomyia species were found in the containers used for plant rooting and ant guards.

Table 2. Association of household characteristics with status of dengue virus infection among participants of household-based cluster investigations conducted in
A total of 588 male and female mosquitoes of all species were collected, of which 311 (53%) were collected indoors (Table 3). Of the 183 females of the two putative vector species, 90.3%   Table 3). None of the Ae. aegypti and Ae. polynesiensis pools were positive for detection of DENV, CHIKV, or ZIKV RNA by RT-PCR.

Molecular epidemiology
Phylogenetic analysis of specimens from dengue case-patients demonstrated that the DENV-2 circulating in American Samoa during the 2016-2018 outbreak clustered with sequences of the Cosmopolitan genotype of DENV-2 and was closely related to recent sequences detected in the Solomon Islands and Papua New Guinea (Fig 2). Estimation of the time of the most recent common ancestor to the sequences obtained in this study suggests that the virus detected in 2017 in American Samoa had been circulating in the region for approximately 2.57 years (95% highest posterior density: 1.37-3.86 years).

Discussion
In this investigation, we utilized household-based cluster investigations to identify factors associated with DENV infection in American Samoa and investigated the presence of DENV in both Ae. aegypti and polynesiensis. Although we were not able to incriminate Ae. polynesiensis  as a DENV vector in urbanized areas, recent febrile illness and presence of a household septic tank were significantly associated with DENV infection. These findings are consistent with the expected clinical manifestations of dengue and findings in other jurisdictions where abundance of Ae. aegypti was associated with the presence of septic tanks [1,37,38]. Significant association of household use of a septic tank with DENV infection is consistent with previous research from other jurisdictions in which septic tanks were identified as sites of prominent production of Ae. aegypti [24,25]. Consequently, messaging to community members during the outbreak emphasized the importance of ensuring that septic tanks are sealed to the environment with netting or sealant that prevents mosquitoes from exiting. However, recent limited investigations demonstrated minimal production of Aedes spp. mosquitoes from septic tanks in one village of American Samoa, but substantial production of Culex PLOS GLOBAL PUBLIC HEALTH quinquefasciatus from some tanks. Moreover, as most septic tanks encountered were functioning improperly and may be present more often in communities with lower socioeconomic status, septic tanks in this jurisdiction may be a marker of communities with more traditional risk factors for DENV transmission [1]. Regardless, the relative contribution of septic tanks and above-ground water containers in American Samoa and elsewhere should be evaluated to further assess their potential as risk factors for DENV infection, including the impact of precluding them from serving as mosquito production sites.
Logistical constraints prevented us from quantifying and identifying pupae found in outdoor containers; however, findings from our outdoor immature survey agreed with what has been reported in Samoa and American Samoa [14,39]. We identified fewer water storage drums than what was found in a similar investigation in 2007 [14], although the difference is likely attributable to variations in availability and quality of municipal water, and economic level of residents between villages sampled in the two studies as opposed to changes within villages between 2007 and 2017. Of note, this investigation identified containers collecting water from faulty plumbing inside homes as important production sites for Stegomyia, a finding that will be useful in future vector control initiatives.
Consistent with previous reports from American Samoa and elsewhere in the South Pacific [14,15,17], we detected Ae. aegypti mosquitoes predominantly indoors and Ae. polynesiensis mosquitoes predominantly outdoors. Aspirations have been reported to collect around 25% of the total indoor population of Ae. aegypti [40], suggesting the true abundance of Stegomyia in American Samoa is higher than what we recorded.
Although age was not significantly associated with DENV infection in this investigation, this may have been a result of the small number of DENV-infected participants identified. Among cases detected during this outbreak, incidence was highest among patients aged <20 years [28]. This observation is consistent with both the expected epidemiology of dengue, as well as a report of DENV-2 having circulated in the Pacific, including the neighboring island nation of Samoa, in 1997 [41]. Although we are unaware of documentation of circulation of DENV-2 in American Samoa in 1997 or soon thereafter, high attack rates in those years would be consistent with susceptibility of residents aged <20 years during the outbreak of DENV-2 during 2016-2018.
The molecular epidemiology of the DENV-2 circulating during the 2016-2018 dengue outbreak in American Samoa demonstrated that the closest relative was an isolate from Solomon Islands in 2016, and that divergence from the most recent known common ancestor occurred roughly two years prior. Both of these findings fit well with the index case-patient of the 2016-2018 outbreak being a fisherman who arrived in American Samoa in November 2016 one week after departing the Solomon Islands [28].
This investigation was subject to several limitations. First, a small (4%) proportion of household-based cluster investigation participants had evidence of acute or recent DENV infection, which decreased the power of the investigation to identify risk factors associated with infection. This small proportion may be attributable to the cluster investigations having been conducted relatively early in the course of the outbreak when only~25% of all cases detected during the outbreak had been identified. Second, because neither the households nor the individuals that participated in this investigation were randomly selected, and because adults more often participated in investigations than children, the observed incidence of DENV infection should not be considered representative of all residents of American Samoa, neither at the time of the survey nor throughout the duration of the outbreak. Similarly, because this investigation was conducted during a public health response and was not designed to be representative of all residents of American Samoa, the findings should not be considered to be generalizable. Last, although questionnaires were administered in both English and Samoan, only a standardized English version was available. As a result, interviewers translated questions during the interview, which may have resulted in variability in how questions were asked and consequent differences in interpretation of some questions by participants.
In conclusion, this report identifies septic tanks as an important risk factor associated with DENV infection in American Samoa. Further study is needed to determine whether septic tanks in American Samoa are significant sources of mosquito vectors of DENV or if the observed association between use of septic tanks and incidence of dengue is due to other factors. In many cases, interventions to block mosquito access to septic tanks could be implemented quickly, easily, and at relatively low cost. These findings will be of importance in the likely event of further dengue outbreaks in American Samoa and elsewhere in the Pacific. Similarly, although we confirmed that Ae. aegypti were more frequently present indoors whereas Ae. polynesiensis were predominantly encountered outdoors, future efforts should more systematically and longitudinally collect specimens to investigate the respective roles of Ae. aegypti and Ae. polynesiensis in transmitting DENV, CHIKV, and ZIKV.